WO1995007096A1 - Traitement de maladies auto-immunes - Google Patents

Traitement de maladies auto-immunes Download PDF

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Publication number
WO1995007096A1
WO1995007096A1 PCT/AU1994/000522 AU9400522W WO9507096A1 WO 1995007096 A1 WO1995007096 A1 WO 1995007096A1 AU 9400522 W AU9400522 W AU 9400522W WO 9507096 A1 WO9507096 A1 WO 9507096A1
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WIPO (PCT)
Prior art keywords
mog
peptide
protein
human
analogue
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PCT/AU1994/000522
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English (en)
Inventor
Claude Charles Andre Bernard
Nicole Claude Marie Kerlero De Rosbo
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La Trobe University
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Publication date
Application filed by La Trobe University filed Critical La Trobe University
Priority to AU76469/94A priority Critical patent/AU7646994A/en
Publication of WO1995007096A1 publication Critical patent/WO1995007096A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to therapeutic and
  • autoantigen which is specific for the autoimmune disease, or an anti-idiotypic antibody thereto, or monoclonal anti-idiotypic antibodies thereto.
  • the present invention relates to a method of, and pharmaceutically-acceptable/veterinary-acceptable compositions suitable for, treating the T-cell and/or
  • MOG myelin oligodendrocyte glycoprotein
  • MOG-like protein a so-called "MOG-like protein”
  • an "immunodominant region" of MOG or MOG-like protein in particular, an "immunodominant
  • the present invention also embraces diagnostic reagents, methods and kits for detecting multiple sclerosis or other T-cell and/or B-cell mediated autoimmune diseases in humans or lower animals.
  • the present invention further embraces nucleotide
  • sequences in particular, such sequences that code for human MOG or human MOG-like protein or an immunodominant region of human MOG or an immunodominant epitope of human MOG, including peptides of amino acids 35-55 of human MOG or amino acids 37-44 of human MOG or amino acids 44-53 of human MOG or analogues of any such peptides, as well as embracing human MOG or human MOG-like protein or an immunodominant region of human MOG or an immunodominant epitope of human MOG, including peptides of amino acids 35-55 of human MOG or amino acids 37-44 of hurr.an MOG or amino acids 44-53 of human MOG or analogues of any such peptides, obtained by standard procedures for protein synthesis or by recombinant DNA techniques.
  • the present invention still further embraces cDNA that encodes for human MOG, as well as cDNA that encodes for an isoform of human MOG .
  • MS Multiple sclerosis
  • CNS central nervous system
  • MS is an inflammatory disease of the central nervous system (CNS) characterised by myelin destruction and a reduced number of oligodendrocytes, the cells that synthesise and maintain myelin within the CNS.
  • MS is thought to be an autoimmune disease.
  • CNS central nervous system
  • macrophages In the CNS of MS patients, there is an accumulation of macrophages, plasma cells, antigen-presenting cells and cytokine-secreting T lymphocytes.
  • the activated T-cells in the brain lesions of individuals with MS are likely to be critical in the pathology of this disease.
  • this disease is believed to be caused by self-destructive immune effects when T-cells, key agents of the bodies defences against infection, are misdirected into immunity towards normal constituents of the nervous system of the individual.
  • EAE autoimmune encephalomyelitis
  • CREA chronic relapsing form of EAE
  • myelin basic protein (MBP) and proteolipid protein (PLP) are the major myelin proteins
  • encephalitogens present in CNS tissue which are responsible for eliciting EAE. Indeed, purified MBP or PLP and
  • MOG is directly accessible to an immune response within the CNS, making it a prime candidate target for antibody-mediated demyelination.
  • MOG-like protein means sequences of amino acids forming a peptide that has substantially the same biological activity as myelin oligodendrocyte
  • glycoprotein in suppressing or eliminating T-cell and/or B-cell mediated or T- and B-cell dependent autoimmune response in an animal such sequences embracing amino acid sequences corresponding to those of myelin oligodendrocyte glycoprotein of the animal except for the omission or addition or substitution or rearrangement of any
  • substitution or rearrangement does not significantly affect the activity of the peptide in suppressing or eliminating T-cell and/or B-cell mediated or T- and B- cell dependent autoimmune response in the animal.
  • an analogue of that peptide as used herein in relation to the peptide of amino acids 35-55 of MOG, or the peptide of amino acids 37-44 of MOG, or the peptide of amino acids 44-53 of MOG, means sequences of amino acids forming a peptide that has substantially the same biological activity as the peptide of amino acids 35-55, or of amino acids 37-44, or of amino acids 44-53,
  • myelin oligodendrocyte glycoprorein m suppressing or eliminating T-cell and/or B-cell mediated or T-and B-cell dependent autoimmune response in an animal
  • substitution or rearrangement does not significantly affect the activity of the peptide in suppressing or eliminating T-cell and/or B-cell mediated or T- and B- cell dependent autoimmune response in the animal.
  • immunodominant epitope as used herein m
  • immunodominant region as used herein in relation to MOG or MOG-like protein, means a region of MOG or MOG-like protein containing an immunodominant epitope.
  • epitopes of MOG or MOG-like protein are species-specifIC, that is, the immunodominant regions/immunodominant epitopes of MOG or MOG-like protein vary between species.
  • anti-idiotypic antibody m relation to an antibody to MOG or MOG-like protein or an immunodominant region/immunodommant epitope of MOG or MOG-like protein as used herein, means an antibody or fragment thereof that reacts with an antibody to MOG or MOG-like protein or an immunodominant region/immunodominant epitope of MOG or MOG-like protein, or to a fragment of such an antibody to MOG or MOG-like protein or an immunodominant region/immuno-dominant epitope of MOG or MOG-like protein.
  • MOG-like protein as used herein, means a monoclonal antibody/ antibodies or fragment thereof that reacts with an antibody to MOG or MOG-like protein or an immunodominant region/ immunodominant epitope of MOG or MOG-like protein, or to a fragment of such an antibody to MOG or MOG-like protein or an immunodominant region/immunodominant epitope of MOG or MOG-like protein.
  • another putative autoantigen means any substance normally present in an animal that is not recognised as normally part of the animal by the lymphocytes or antibodies of the animal, resulting in the substance being attacked by the immunoregulatory system of the animal as though the substance was foreign to the animal.
  • a fragment of another putative autoantigen means a portion of another putative autoantigen that
  • an analogue of another putative autoantigen means compounds so chemically or structurally similar to the putative autoantigen that those compounds have substantially the same biological activity as the putative autoantigens, and elicit an attack by the immunoregulatory system of the animal equivalent to that elicited by the putative autoantigen.
  • a fragmant of an analogue of another putative autoantigen means a portion of an analogue of another putative autoantigen that possesses substantially the same biological and immunological activity as the analogue but need not possess the autoantigenic properties of the entire analogue.
  • the present invention provides
  • T-cell and/or B-cell mediated autoimmune disease in humans or lower animals, comprising the administration to the human or animal, of an effective amount of at least one active agent selected from:
  • MOG-like protein or an immunodominant region of MOG or MOG-like protein, in particular, an immunodominant epitope of MOG or MOG-like protein, including:
  • autoantigen(s) a fragment(s) of an analogue of another putative autoantigen(s); another therapeutic or prophylactic agent(s); a pharmaceutically-acceptable or veterinary-acceptable carrier(s) or adjuvant(s).
  • the present invention provides
  • the present invention provides the use of at least one active agent selected from item (i) to item (iii) of the method that constitutes the first aspect of the present invention, for the manufacture of a
  • compositions or veterinary composition suitable for the therapeutic or prophylactic treatment of a T-cell and/or B-cell mediated autoimmune disease in humans or lower animals, respectively.
  • the present invention provides
  • nucleotide sequences substantially in accordance with: the nucleotide sequence -114 to 1774 of Figure 7A herein, which includes the 3' and 5' untranslated regions of the gene of human MOG; or the nucleotide sequence 1 to 741 of Figure 7A herein, which comprises the translated region of the gene of human MOG; or the nucleotide sequence -114 to 1603 of
  • Figure 7B which includes the 3' and 5' untranslated regions of the gene of a truncated form of human MOG; or the nucleotide sequence 1 to 609 of Figure 7B herein, which comprises the translated region of the gene of a truncated form of human MOG; or the nucleotide sequence 1 to 450 or 1 to 741 or 190 to 252 of Figure 7A herein, which comprise immunodominant regions/epitopes of the translated region of the gene of human MOG.
  • This fourth aspect of the present invention also includes a cDNA that encodes the 24,000 Mr human MOG, as well as a cDNA that encodes an isoform of MOG truncated to 174 amino acids.
  • the present invention provides an amino acid sequence or protein substantially in accordance with Figure 7A or Figure 9 herein, as well as the human MOG amino acid sequence in accordance with Figure 8 or 10; or corresponding to the nucleotide sequence of the fourth aspect of the present invention.
  • the present invention provides a method of isolation and purification of MOG from central nervous system white matter using immunoaffinity chromotography so as to obtain MOG devoid of other major myelin proteins and in quantities sufficient for further investigations of its encephalitogenic and immunogenic properties, said method comprising: the isolation of membrane proteins from an homogenate of the white matter; the extraction from the membrane proteins by deoxycholate of a soluble fraction containing MOG; and the isolation of MOG from that soluble fraction by immunoaffinity chromatography on an anti-MOG IgG column, the MOG being eluted, in the absence of detergent, with 50 mM diethylamine pH 11.5, which was then dialysed out or removed on a desalting column.
  • the present invention provides anti-idiotypic antibodies or monoclonal anti-idiotypic
  • the present invention provides diagnostic reagents which comprise an anti-idiotypic antibody or monoclonal anti-idiotypic antibodies according to the seventh aspect of the present invention, which is labelled so as to permit detection of the reaction between the anti-idiotypic antibody or monoclonal anti-idiotypic antibodies, with the antibody to an active agent selected from item (i) of the method that constitutes the first aspect of the invention.
  • the present invention provides methods of detecting an antibody to an active agent selected from item (i) of the method that constitutes the first aspect of the present invention in a sample, including a tissue or blood or serum or cerebrospinal fluid (CSF) or urine sample or fraction thereof, taken from a human or a lower animal, which method comprises contacting said sample with an anti-idiotypic antibody or monoclonal anti-idiotypic antibodies of the seventh aspect of the invention, or a diagnostic reagent of the eighth aspect of the invention, and detecting binding of said antibody to said anti-idiotypic antibody or said monoclonal anti-idiotypic antibodies or said diagnostic reagent to indicate the presence of said antibody in said sample.
  • a sample including a tissue or blood or serum or cerebrospinal fluid (CSF) or urine sample or fraction thereof
  • the present invention provides methods of detecting a T-cell and/or B-cell mediated autoimmune disease in humans or lower animals, including the detection of multiple sclerosis in a patient, comprising determining the level or amount of an antibody to at least one active agent selected from item (i) of the method that constitutes the first aspect of the present invention, m a sample, including a tissue or blood or serum or CSF or urine sample or fraction thereof, taken from the human or lower animal, and optionally comparing the level or amount so obtained with that obtained from a sample taken from a human or lower animal not having multiple sclerosis or another T-cell and/or B-cell mediated autoimmune disease.
  • the antibody to at least one active agent selected from item (i) of the method that constitutes the first aspect of the present invention may be detected or determined with a labelled agent selected from item (ii) and (in) of the method that constitutes the first aspect of the present invention.
  • multiple sclerosis or another T-cell and/or B-cell mediated autoimmune disease in humans or lower animals may be detected by determining the level or extent of degradation of myelin or a component thereof, in particular, myelin basic protein (MBP), following upon incubation of the myelin or component thereof with sera or an immunoglobulin or other fraction of the human or animal containing an antibody to an active agent selected from item (i) of the method that constitutes the first aspect of the present invention, and optionally comparing the level or extent of the degradation of myelin or component thereof so obtained with that obtained from sera or an immunoglobulin or other fraction of a human or lower animal not having multiple sclerosis or another T-cell and/or B-cell mediated autoimmune disease.
  • MBP myelin basic protein
  • the present invention provides diagnostic test kits suitable for detecting a T-cell and/or B-cell mediated autoimmune disease in humans or lower animals, including the detection of multiple sclerosis in a patient, comprising a receptacle for receiving and retaining a sample including a tissue or blood or serum or CSF or urine sample or fraction thereof, taken from the human or lower animal, and a detection means for communicating with the sample, which detection means comprises: a reactive agent selected from item (ii) or (iii) of the method that constitutes the first aspect of the present invention, which may be suspended on a carrier; and a reagent(s) that is responsive to a reaction between an antibody of the human or lower animal and the anti-idiotypic antibody or monoclonal anti-idiotypic antibodies selected from said item (ii) or (iii), whereby reaction of the anti-idiotypic antibody or monoclonal anti-idiotypic antibodies with an antibody of the human or lower animal diagnostically indicates a T-cell and/or B-cell
  • the present invention provides methods of detecting MOG or MOG-like protein in a body fluid, including serum, CSF or urine, using antibodies to MOG or MOG-like protein, in particular, polyclonal antibodies to MOG or MOG-like protein, especially labelled polyclonal
  • the active agent is human MOG, or human MOG-like protein, or an immunodominant region or imunodominant epitope of human MOG or human MOG-like protein; or
  • the active agent is the peptide of amino acids 35-55 of human MOG or an analogue of that peptide; or
  • the active agent is the peptide of amino acids 37-44 of human MOG or an analogue of that peptide; or
  • the active agent is the peptide of amino acids 44-53 of human MOG or an analogue of that peptide; or 5.
  • the active agent is an anti-idiotypic antibody to: human MOG; or human MOG-like protein; or a peptide of amino acids 35-55 of human MOG or an analogue of that peptide; or a peptide of amino acids 37-44 of human MOG or an analogue of that peptide; or a peptide of amino acids 44-53 of human MOG or an analogue of that peptide; or
  • the active agent comprises monoclonal anti-idiotypic antibodies to: human MOG; or human MOG-like protein; or a peptide of amino acids 35-55 of human MOG or an analogue of that peptide; or a peptide of amino acids 37-44 of human MOG or an analogue of that peptide; or a peptide of amino acids 44-53 of human MOG or an analogue of that peptide.
  • the active agent is administered to the human or animal in the form of a pharmaceutically-acceptable or veterinary-acceptable composition, respectively, in which the active agent is formulated with respectively a
  • the active agent is administered to a human in association with a pharmaceutically-acceptable carrier or adjuvant;
  • the active agent is orally administered to the human or animal to enable enteral uptake of the active agent;
  • the active agent is intramuscularly administered to the human or animal in the form of an injectible oil-based formulation of the active agent;
  • the active agent is intravenously administered to the human or animal in the form of an injectible saline solution, or balanced salt solution, or other
  • the active agent is administered to the human or animal in an amount effective to induce immunological tolerance to MOG or MOG-like protein; or 7. the active agent is administered to a human being before or after the onset of multiple sclerosis in an amount effective to induce immunological tolerance to MOG or MCG-like protein or to treat multiple sclerosis; or 8. the active agent is administered to the human or animal at dosage rates within the range of 0.1mg to 500mg per day, preferably 0.2mg to 200mg per day, more preferably 0.5mg to 100mg per day.
  • the anti-idiotypic antibody or monoclonal anti-idiotypic antibodies may be used in a variety of ways, for example, they may be used for inhibiting the binding between the antigen and the T-cells and/or B-cells, or for killing the T-cells, or for isolating the T-cells.
  • a toxin or radioactive substituent may be bonded to the anti-idiotypic antibody or monoclonal anti-idiotypic antibodies, which in the case of the bonding of a radioactive substituent results in a lethal dosage of radioactivity upon binding of the anti-idiotypic antibody or monoclonal anti-idiotypic antibodies to the T-cells of the autoimmune disease.
  • the anti-idiotypic antibody or monoclonal anti-idiotypic antibody may be conjugated with an agent for specific elimination of the undesired T-cell.
  • the anti-idiotypic antibody or monoclonal anti-idiotypic antibodies may be formulated with conventional pharmaceutically acceptable adjuvants or diluents, and may be administered by any suitable route, preferably
  • the dosage amount of active agent and frequency of administration which may be for periods of 5-25 years, will depend upon whether the treatment is prophylactic or therapeutic, and in the case of the latter, the severity of the disease and physical condition of the patient, as would be well understood in the art.
  • optimization of dosage levels and frequency is a matter of ordinary practice in treating multiple sclerosis in humans or treating another T-cell and/or B-cell mediated autoimmune disease in humans or lower animals.
  • the active agent which includes MOG or MOG-like protein, especially the peptide of amino acids 35-55 of MOG or an analogue of that peptide, or an anti-idiotypic antibody thereto or monoclonal anti-idiotypic antibodies thereto, may be administered to the animal in various ways such as orally, intravenously, intramuscularly, intraperitoneally, intranasally,
  • the MOG or MOG-like protein is intradermally or subcutaneously.
  • the MOG or MOG-like protein is especially the peptide of amino acids 35-55 of MOG or an analogue of that peptide.
  • the active agent is formulated for oral
  • the active agent is formulated for intramuscular administration to the human or animal in the form of an injectible oil-based formulation of the active agent; or 3. the active agent is formulated for intravenous administration to the human or animal in the form of an injectible saline solution, or balanced salt solution, or other physiologically acceptable water-based formulation of the active agent; or
  • the active agent is formulated in a dosage unit form to be administered to the human or animal at dosage rates within the range of 0.1mg to 500mg per day, preferably
  • the active agent is formulated in association with a pharmaceutically-acceptable carrier or adjuvant.
  • compositions or veterinary compositions of the present invention can be formulated by methods well known for the formulation of pharmaceutically-acceptable or veterinary-acceptable compositions, wherein the active agent, which includes MOG or MOG-like protein, especially the peptide of amino acids 35-55 of MOG or an analogue of that peptide, or an anti-idiotypic antibody thereto or monoclonal anti-idiotypic antibodies thereto, is combined in admixture with a pharmaceutically-acceptable or
  • veterinary-acceptable carrier substance the nature of which usually depends on the particular mode of
  • compositions and formulations which are suitable for delivery of the active agent, which includes MOG or MOG-like protein, especially the peptide of amino acids 35-55 of MOG or an analogue of that peptide, or an tnti-idiotypic antibody thereto or monoclonal anti-idiotypic antibodies thereto, in accordance with the therapeutic or prophylactic method of the present invention.
  • enteral formulations are usually solids, semi-solids, suspensions or emulsions that use pharmaceutically acceptable salts, carriers, flavour enhances, or the like.
  • Parenteral formulations are usually injectable fluids that use physiologically
  • the autoimmune response which underlies multiple sclerosis may be treated by administration of MOG or MOG-like protein, especially the peptide of amino acids 35-55 of MOG or the amino acids 37-44 of MOG or the amino acids 44-53 of MOG or an analogue of any such peptide, or an anti-idiotypic antibody thereto or
  • MS may be effectively suppressed by the oral or enteral administration of MOG or MOG-like protein
  • such administration is per os.
  • MS may be effectively suppressed by administration of MOG or MOG-like protein, especially the peptide of amino acids 35-55 of MOG or the amino acids 37-44 of MOG or the amino acids 44-53 of MOG or an analogue of any such peptide, or an anti-idiotypic antibody thereto or monoclonal anti-idiotypic antibodies thereto, in an amount of from 0.1mg to 500mg per day, and may be
  • the MOG or MOG-like protein is administered orally in an amount of from 0.5mg to 500mg per day.
  • the exact dosage of MOG or MOG-like protein especially the peptide of amino acids 35-55 of MOG or the amino acids 37-44 of MOG or the amino acids 44-53 of MOG or an analogue of any such peptide, or an anti-idiotypic antibody thereto or
  • compositions or veterinary compositions of the present invention is a function of the age, sex, and physical condition of the patient, as well as other concurrent treatments, including other myelin protein (s), being administered.
  • compositions may be administered to an animal in need of treatment for such autoimmune disease so as to
  • Such preparations may also be administered to an animal who is predisposed to developing such autoimmune disease so as to prevent the onset of such disease or to lessen the severity of such disease when it does emerge.
  • the anti-idiotypic antibody or monoclonal anti-idiotypic antibodies is labelled, preferably by horse-radish
  • the sample is a blood or serum or CSF or urine sample
  • the detecting step may comprise immunochemical staining of the sample.
  • the anti-idiotypic antibody or monoclonal anti-idiotypic antibodies is labelled so as to change colour upon reaction with the antibody of the human or lower animal in the sample, more preferably the colour being proportional to the concentration of the antibody of the human or lower animal in said sample.
  • the antibodies in particular, the anti-idiotypic
  • antibodies or monclonal anti-idiotypic antibodies, of the present invention, as well as the diagnostic agents of the present invention may be prepared as described herein or by known methods.
  • the nucleotide sequences of the present invention may be expressed by known methods.
  • CNS component quantitatively minor CNS component, is one such externally located antigen, which we postulate to be the putative target antigen in the autoantibody mediated demyelinating pathway in MS.
  • immune responses to MOG may initiate autoimmune mediated demyelination, we have assessed the T-cell proliferative response, as well as the B-cell response, to highly purified MOG by peripheral blood lymphocytes from MS patients and ascertained the
  • the final product which migrated as two bands of molecular weight 28 kDa and 58 kDa, was highly pure as indicated by specific reactivity with monoclonal anti-MOG antibodies on immunoblots in the absence of any detectable reactivity with antibodies specific for MBP, PLP and myelin-associated glycoprotein (MAG).
  • MAG myelin-associated glycoprotein
  • HNK-1 epitope which is also found in other human myelin glycoproteins such as MAG and PO. This carbonydrate epitope is believed to be of importance in cell-cell adhesion, making it an important part of the molecule which should be considered m the context of autoimmune demyelination.
  • Partial ammo acid sequence was obtained from both MOG bands separated by SDS-PAGE and electroblotted onto polyvmylidene difluoride membrane. The sequence of the first 18 N-terminal ammo acids has approximately 55% homology with the reported rat MOG sequence deduced from the cloned cDNA sequence; small internal sequences also obtained, showed very high homology.
  • MOG as a primary target for the initiation of autoimmune-mediated demyelination, by injecting Lewis rats in the hind foot pads with highly purified MOG emulsified in complete Freund's adjuvant (CFA) supplemented with Mycobacte ⁇ um tuberculosis (Bernard et al., 1976).
  • CFA complete Freund's adjuvant
  • Mycobacte ⁇ um tuberculosis Mycobacte ⁇ um tuberculosis
  • myelin was purified from human brain as previously described (Norton and
  • rat Igs were purified using a DEAE Affi-Gel blue-sepnarose column (Biorad) according to the
  • myelin incubations and quantification of MBP degradation were as described previously (Kerlero de Rosbo and Bernard, 1989), incubation mixtures were prepared with 200ug of myelin protein m 150 ul of 100 mM Tris/ acetic acid pH 7.5 m the presence of serum Igs (25 ug) or the anti-MOG monoclonal antibody
  • FIG 1 shows the MBP degradation in human myelin
  • Igs lmmunoglobulins isolated from sera of affected rats.
  • Each column represents the mean + SEM of percent MBP decrease in five separate myelin preparations incubated individually with Igs from control rats injected with CFA, with monoclonal anti- MOG Igs (8-18C5), with MOG-EAE Igs (from two
  • demyelination together with a monospecific humoral immune response directed to the encephalitogenic molecule in the absence of reactivity to other known CNS proteins.
  • MOG is the predominant myelin antigen recognised by peripheral blood lymphocytes of MS patients (Kerlero de Rosbo et al, 1993), we believe that the immune response to MOG in MS is of importance in the pathogenesis of the disease. Whilst it is now well established that MOG is capable of
  • the purification procedure comprises: the isolation of membrane proteins from an homogenate of CNS white matter; the extraction from the membrane proteins by deoxycholate of a soluble fraction containing MOG, and the isolation of MOG from that soluble fraction by
  • the beads were then poured into a column and equilibrated with 25 mM Tris/HCI pH 8.0 for 30 mins. MOG was eluted with 50 mM diethylamine in 25 mM Tris/HCI, pH 11.5.
  • Sepharose 4 Fast Flow Total proteins were determined according to Lowry et al. (1951). MATERIALS AND METHODS
  • CNS tissues Post-mortem CNS tissues were obtained at autopsy of patients who had died of non-neurological causes; the white matter was dissected out on ice, frozen in liquid nitrogen and stored at -80°C.
  • Human myelin from CNS white matter was purified by the method of Norton and Poduslo (1973).
  • Human myelin basic protein (MBP) was purified from CNS white matter as described by Dunkley and Carnegie (1974). Such a
  • Rabbit polyclonal antiserum raised against the sequence encompassing amino acid residues 117-129 of the proteolipid protein (PLP) (Trifilieff et al., 1986) was obtained from Dr. E. Trifilieff, Centre de Neurochimie du CNRS, France, France.
  • Rabbit polyclonal anti-mouse IgM (Cat. No. 64-365-1) and anti-mouse IgG (Cat. No. 65-125-1) antisera were obtained from ICN Biomedicals (NSW,
  • Rabbit polyclonal anti-human polyvalent immunoglobulins antiserum (Cat. No. 1-8010) was obtained from Sigma, USA. Mouse monoclonal antibody raised against a plant glycoprotein was obtained from Dr. P. Meikle, Biochemistry Dept, La Trobe University. Mouse monoclonal anti-MOG antibody, clone 8-18C5 (Linington et al., 1984), was purified from ascites fluid by affinity chromatography on a Protein G Sepharose 4 Fast Flow column (Pharmacia LKB, Uppsala, Sweden; Cat. No. 17-0618-01) according to the manufacturer's specifications.
  • Protein A as the visualizing agent (Kerlero de Rosbo et al., 1984) except that polyvinylidene difluoride membrane (PVDF) (Immunobilon-P, Millipore, Bedford, MA, USA: Cat. No. IPVH 00010), rather than nitrocellulose, was used.
  • PVDF polyvinylidene difluoride membrane
  • electrophoresis After electrophoresis, the gel was equilibrated in 10mM CAPS, 10% methanol, pH 11.0 containing ImM thioglycollic acid for 30 mins prior to electroblotting onto PVDF membrane.
  • Electrotransfer was performed using a Bio-Rad Transblot apparatus at 90V for 4 hours at 4°C. Protein as
  • HCO 3 containing 0.02% Tween-20 and 2 ug of modified trypsin. Following proteolytic digestion for 12 hrs at 37°C, the peptides were extracted with the following series of solvents: 1% (4 hrs), 70% TFA (2 ⁇ 4 hrs), 50% TFA, 50% acetonitrile (2 ⁇ 4 hrs). Tween-20 (0.02%) was included in all solvents. The combined eluates were then evaporated to near dryness by centrifugal lyophilisation prior to purification of peptides by RP-HPLC.
  • Microbore column RP-HPLC purification of peptides was performed using a Hewlett-Packard (Waldbronn, FRG) liquid chromatograph (model 1090A) equipped with a diode-array detector (model 1040A). Peptides were separated on a
  • Brownlee RP-300 column (30mm ⁇ 2.1mm I.D., Applied Biosystems CA, USA) developed with a linear 60 minute gradient from 100%A to 100%B.
  • Solvent A was 0.1% (v/v) aqueous trifluoroacetic acid and solvent B was 60%
  • Fig. 2 represents the electrophoretic profile of purified human MOG showing 2 major bands which migrate with approximate molecular weights of 28 kDa and 58 kDa respectively; both bands reacted with the monoclonal anti-MOG antibody 8-18C5; an additional band of molecular weight 35 kDa detected with 8-18C5 upon immunoblotting was seen infrequently on the gels.
  • Rat MOG isolated by the same procedure comprised a doublet of molecular weight 52/58 kDa and a single protein of molecular weight 28 kDa (data not shown); all three rat bands also reacted with 8-18C5.
  • MOG eluted from the anti-MOG IgG affinity column was immunoblotted with mouse monoclonal anti-MOG antibody which requires a secondary rabbit anti-mouse IgG antibody to allow binding, and thereby visualization of the antigen-antibody complex, by 175I Protein
  • Control immunoprobing of the protein originally electrophoresed under reducing conditions comprised an incubation with an irrelevant primary mouse monoclonal antibody (monoclonal anti-plant
  • the carbohydrate moiety of a subpopulation of human MOG contains the HNK-1 epitope. No staining of MOG with anti-HNK-1 antibody was detected in whole myelin; this is due not only to the fact that MOG is present in very small quantities in myelin, but also to the fact that the carbohydrate moiety of MOG represents only approximately 5-10% of the whole molecule.
  • Figure 7 herein sets out the nucleotide sequence of human MOG as determined by us, as well as setting out the deduced amino acid sequence of human MOG, whilst Figure 8 herein sets out an amino acid comparison between human MOG and rat MOG, whilst Figure 9 herein sets out the amino acid
  • RNA was extracted from rat brain tissue by the acid guanidinium thiocynate-phenol-chloroform method
  • RNA was reverse transcribed into 1st-strand cDNA in a reaction consisting of 1 x Gene Amp PCR buffer (Perkin Elmer), 0.25mM of each dNTP (Perkin Elmer), 0.05ug/ul random hexamers (Pharmacia), 0.5U/ul RNasin (Promega) and 10U/ul Superscript RNase
  • H--reverse transcriptase (Gibco BRL). The reaction mix was incubated 10 min at room temperature, then 45 min at 42°C. The enzyme was inactivated by heating at 95°C for 5 min and the reaction chilled on ice prior to
  • PCR polymerase chain reaction
  • a lambda gt10 human adult medulla cDNA library (Clontech #HL1089a) was plated at a density of 50,000 plaques/150mm plate. Duplicate lifts were taken using Colony Plaque Screen Membranes (Dupont) and processed according to the manufacturer's instructions. After pre-hybridisation of the filters, the rat MOG probe was oligolabelled with a-dCTP 32 (Amersham Multiprime Labelling System) and hybridised to the membranes for 16 h at 42°C at a concentration of 5 x
  • the hybridisation solution contained 50%
  • Lambda DNA was purified from phage lysate using MAGIC Lambda Preps DNA Purification System (Promega) and the clones were sequenced directly, without sub-cloning, by cycle sequencing using Promega fmol DNA sequencing system as detailed in the manufacturer's instructions. The sequence of several human MOG cDNA clones was determined in both directions. The sequences were assembled by the AssemblyLIGN program (IBI) and analysed by Mac Vector (IBI) and BLAST programs.
  • RTPCR Reverse transcription PCR
  • RTPCR method was identical to that used to amplify the rat MOG probe.
  • the RTPCR was performed on MS and control white matter RNA.
  • primers were chosen to sequence into the introns from the genomic exon sequence. Due to the size of several of the exons a sub-genomic library was constructed in order to sequence both donor and acceptor splice sites of the boundaries.
  • MOG genomic clones that contained the relevant exons were digested to completion using the restriction enzyme Sau 3A.
  • the digestion products were ligated overnight with a pUC18 vector cut with BamH I, transformed and replica plated on colony plaque screen membranes (Dupont) and processed according to the manufacturer's instructions.
  • ECL 3'-oligolabelling and detection system oligo- nucleotides specific for each exon were used to screen the sub-genomic library and clones containing the regions of interest were isolated.
  • DNA was prepared for sequencing using alkaline lysis minipreparation method (Sambrook et al., 1989).
  • RNA was extracted from control white matter and poly A + RNA was purified using a mRNA purification kit
  • xug of mRNA was fractioned on a 1% agarose/2.2M formaldehyde gel (Sambrook et al., 1989) and transferred to Hybond-N+membrane.
  • the northern filter was probed with the coding region of MOG using the same probe used to screen the genomic library.
  • RNAse H is an enzyme which cleaves the RNA strand of RNA/DNA heteroduplexes and annealing of an oligonucleotide or DNA fragment to complementary regions of RNA molecule enables cleavage of the mRNA in a site specific manner (Leis et al., 1973; Donis-Keller, 1979). Total RNA was extracted from MS and control white matter and the poly A + RNA was purified.
  • RNAse H mapping 8ug of poly A + RNA was co-precipitated with 80 pmol of the oligonucleotide 6f, a 25mer complementary to an internal sequence of MOG (Table 7). The pellet was resuspended in 20ul RNAse H buffer (20mM Tris pH 7.5, 10mM MgCl 2 , 100mM KC1, 0.1mM dithiothrietol, 5% sucrose). After 10 min at 70°C the sample was hybridised for 30 min at
  • RNAse H (Pharmacia) was added to the test sample and digested at 37°C for 45 min. An undigested control was used for each experiment. RNA was precipitated and
  • RNA loading buffer RNA loading buffer. Digested and undigested samples were fractioned by conventional northern technique, transferred and probed with fragments 5' and 3' of the digestion site to determine the size and number of
  • the 5' probe (probe 1) was
  • the primers 9f and 9(3') were used to amplify a 3' probe that excluded the truncated form of MOG (probe 2) and a probe specific for truncated MOG was amplified using the primers 3a and 3g (probe 3) (Table 7).
  • the probe used for the MOG genomic library screen was also used for the southern hybridisation. Filters were
  • a 970 bp Eco RI fragment containing the entire coding region of human MOG and a 730 bp Eco RI/Dra I fragment containing the coding region of the truncated form of MOG was ligated to adaptors and subcloned into the Bst XI site of the mammalian expression vector pEF-BOS (Mizushima and Nagata, 1990).
  • Proteins were then transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore) at 100V for 1 h in 192mM glycine, 20% (v/v) methanol and 25mM tris-Cl, pH8.3.
  • Immobilon-P Millipore
  • the coding rection of rat MOG was amplified using RTPCR.
  • the 744 bp product was purified and used as a probe to screen a human medulla cDNA library. From this library eleven clones were plaque purified by successive rounds of screening and sequenced on both strands using oligonucleotide primers
  • Human MOG sequence A majority of the human MOG clones analysed encoded a full length 'MOG' protein, however, nearly a quearter of MOG clones analysed appeared to encode an alternative form of the protein.
  • the major human MOG cDNA species has an open reading from of 741 bp encoding a predicted polypeptide containing a signal peptide of 29 amino acids followed by 218 amino acids of mature protein.
  • Human MOG contains an Ig-like domain, two putative membrane spanning regions and a
  • N-linked glycosylation site at Asn-31 The clones sequenced had a 5' untranslated region (UTR)of 116bp and the 3' UTR contained a canonical AATAAA polyadenylation signal 810bp downstream of the stop codon. No polyadenylated clones were isolated.
  • the sequence of the alternate cDNA diverges at amino acid residue 169, resulting in a short stretch of different amino acids followed by an in-frame stop codon, prior to the second transmembrane domain. This species of CDNA is predicted to encode a smaller protein of 174 amino acids.
  • constructs containing the coding regions from the full length and 'truncated' human MOG cDNAs were generated in the mammalian expression vector pEF-BOS.
  • COS cells were
  • 8-18C5 recognises an epitope on the Ig-like domain of the molecule and would be expected to recognise the truncated form of MOG.
  • Genomic clones encoding human MOG were isolated by screening a genomic library with the coding region of human MOG cDNA. We determined that human MOG is encoded by seven exons. The splice donor and splice acceptor sites were sequenced for each intron-exon boundary ( Figure 11). The first exon encodes the 5'-untranslated region and the signal peptide, the second exon encodes the Ig-like domain and the remaining portion of the molecule is encoded by five short exons.
  • the genomic structure of MOG is consistent with the theory that butyrophilin and other members of the Ig sub-family are evolutionally related to MOG through an exon shuffling event of the Ig-like region (Vernet et al., 1994).
  • the truncated form of MOG described here appears to be generated from a failure to splice out of the fourth intron.
  • the splice donor and splice acceptor GT and AG sequences appear to be intact.
  • the splice acceptor site for the intron does lacks the C/T stretch found in the loose splice consensus sequence (C/T) 10 NC/TAG.
  • RNAse-H mapping was performed on MS and control samples. Two RNA species of 1.95 and 1.25kb, were detected without RNAse-H. Following digestion and hybridisation with probe 1 only one fragment of 0.42kb was detectable, indicating that there are no alternative 5' variants. Hybridisation with probe 2, which excludes the truncated form, clearly reveals two bands at 1.5kb and 0.88kb in the digested RNA.
  • the two transcripts observed in the undigested RNA are the result of either differential exon splicing or usage of an alternate polyadenylation signal at the 3' end of the mRNA encoding the full length MOG.
  • the smaller message detected by hybridisation did not appear to correspond to the cDNA variant encoding the truncated protein since it was not detectable by hybridisation with probe 3 (data not shown).
  • the 5' UTR of full length MOG can be estimated to be 100bp and the 3' UTRs, including an unknown length of poly(A) tact, can be calculated to be 490bp and 1100bp. The longer and more abundant UTR would appear to correspond to the
  • polyadenylation signal at position 1554 nt if a poly (A) 'tail' of approximately 250bp long is included (Wickens, 1990). This polyadenylation site is also in a position homologous to the poly(A) signal seen in bovine MOG. The shorter UTR cannot be accounted for and despite analyses of may 3' sequences from the library we could not confirm the use of alternative exons or alternative polyadenylation signals.
  • Human MOG is a 218 amino acid protein containing an Ig-like variable domain and two transmembrane regions and is considered to be a member of the Ig supergene family.
  • the Ig-like variable region domain of MOG appears to be encoded by one exon and the remainder of MOG is encoded by a series of relatively short exons.
  • MOG protein may have at least two isoforms.
  • MOG protein may have at least two isoforms.
  • mRNA species with retained introns are usually the result of mutations in the splice donor or splice acceptor sites (Stover et al, 1993; Su and Lin, 1990), however, in this instance it is not clear what has caused the splicing defect.
  • the presence of a retained intron can affect RNA processing to such a degree that the variant mRNA does not produce a protein (Stover et al,
  • RNAse H mapping also supported the evidence of there being mRNA isoforms of MOG.
  • MOG mRNA isoforms There are at least three MOG mRNA isoforms, the 1.95kb major species that encodes full length MOG, a 1.25kb species with a shorter UTR that may also encode full length MOG, and a mRNA of unknown size that contains a retained intron.
  • the 1.25kb form has not been cloned but its existence is deduced from RNAse H analysis.
  • Human MOG unlike other species of MOG, appears to have a classical polyadenylation signal AATAAA at 1554bp and a non-canonical AATTAA signal overlapping. The presence of the more conventional polyadenylation signal may indicate that human MOG is more efficiently polyadenylated than in other species cloned and could have implications for the half-life of the mRNA.
  • MOG is highly conserved between species, at both the
  • the second transmembrane region in contrasts, is absolutely conserved between species and suggests this portion of MOG may be important in interacting with other molecules in the myelin membrane.
  • the expression of MOG in its native form in the COS cell system will be invaluable to study the structure and function of the MOG protein. High conservation of amino acids implies that all myelin proteins, including MOG, have strict structural requirements that are closely linked to their capacity to function in the myelin sheath.
  • Point mutations, deletions and inversions within the myelin genes such as, PLP, MBP, and MAG have given rise to the myelin deficient mouse strains jimpy, msd, mid, shiverer and quaking (reviewed by Stoffer, 1990) and it could be predicted that mutations of the MOG gene in mice may have a significant phenotype and have a detrimental effect in humans.
  • MS is presumed to be a multi factorial autoimmune disease. Certain Class II HLA variants and T cell receptor alpha chain genes have been reported to be associated with MS
  • polymorphism is not strictly limited to MS as is is also present in some of the healthy controls tested. If the linkage that we have observed could be confirmed or
  • MAG and MOG constitute only approx. 1% and 0.05% of CNS myelin proteins respectively. Because of the low abundance of MAG and MOG in CNS myelin, it is difficult to obtain MAG and MOG in a highly purified form and in sufficient quantities, hence the paucity of reports investigating T cell reactivity to MAG and MOG.
  • Human MBP again was purified from neurologically normal human brain according to the procedure of Dunkley and Carnegie (1974).
  • Human MOG was purified from neurologically normal brain white matter or spinal cord according to the procedure detailed above, in which membrane proteins were extracted by addition of deoxycholate to human white matter
  • MOG homogenate and MOG was isolated from the solubilized protein fraction by immunoaffinity chromatography on an anti-MOG IgG column, the MOG being eluted, in the absence of detergent, with 50 mM diethylamine pH 11.5, which was then dialysed out or removed on a desalting column.
  • Human MAG was purified as described previously by Berger et al (1990). PLP was obtained from a washed total lipid extract of bovine white matter according to procedure of Folch et al (1957), and lipid was removed by chromatography on a
  • the blocking solution consisted of phosphate buffered saline containing 2% non fat dry milk and the hybridoma
  • the second antibody was goat anti-rat IgG conjugated to alkaline phosphatase (Fisher Scientific Co.) and a BioRad alkaline phosphatase conjagate substrate kit was used for detection.
  • Mouse monoclonal anti-rat MOG antibody (clone 8-18C5;17) was purified from ascites fluid by affinity chromatography on a protein G Sepharose 4 Fast Flow column (Pharmacia LKB, Uppsala, Sweden; Cat# 17-0618-01) according to the
  • the cells producing 8-18C5 antibody were obtained from Dr. M. Webb.
  • Mouse monoclonal anti-human MBP antibody (clone 65 (2-1)) was obtained from Prof. P.R. Carnegie, Murdoch University, Western Australia, and purified by chromatography on protein G Sepharose 4 Fast Flow.
  • Mouse monoclonal anti-HNK1 antibody (clone 6-3-19-1; Cat # MON 1010) was purchased from Sanbio
  • Rat monoclonal anti-PLP antibody (clone AB3) was obtained as described by Yamamura et al (1991).
  • Polyclonal anti-PLP antibodies were obtained from Prof. W.B. Macklin, UCLA, Los Angeles, USA and Dr. E. Trifilieff, Centre de Neurochimie du CNRS, France. All 3 antibody preparations were used at various times to assess possible contamination of the MOG preparations with PLP.
  • Venous heparinized blood samples were obtained from
  • the MS group was comprised of 22 consecutive out-patients and 2 in-patients (18 females, 6 males) who agreed upon request to be tested and were not under steroid treatment or other medication known to affect the immune system for at least 1 month prior to the time of testing. Twenty-one patients had definite MS and 3 had probable MS and their Kurtzke's Expanded Disability Status Scale (EDSS) ranged from 2 to 8 (Table 3). The 16 patients and healthy individuals which comprised the control group (9 females, 7 males) were not under medications known to affect the immune system. None of the patients had other inflammatory disease of the CNS.
  • EDSS Kurtzke's Expanded Disability Status Scale
  • PBLs were separated from 20 mls of MS or control blood using Leucosep tubes as described by the manufacturer
  • Isolated PBLs were cultured in microtiter wells in the absence or presence of the relevant antigens (5-25 ug/ml), essentially as described previously by Ben and Cohen (1982). Briefly,
  • thymidine incorporation expressed as mean counts per minute (cpm) of triplicate cultures.
  • Stimulation indices are calculated as proliferative response in the presence of antigen, divided by proliferative response in the absence of antigen. In view of the difficulties encountered to purify adequate quantities of MOG suitable for cell culture, not all lymphocyte samples could be tested at different concentrations of this antigen.
  • Anti-HNK1 antibody- reactive bands could not be detected in MOG preparations at molecular weights for MOG (90-100 kDa); however, such reactivity was observed for bands
  • peripheral blood lymphocyte sensitization to MBP could be detected in some MS patients, but also in patients with other neurological diseases and in healthy controls.
  • MOG is a primary target for autoimmune responses associated with demyelinating diseases
  • MOG or MOG-like protein or immunodominant region/epitope thereof may be administered to the animal by: a) subcutaneous injection in incomplete Freund's adjuvant (IFA); b) intra-peritoneal injection; or c) oral administration.
  • IFA incomplete Freund's adjuvant
  • An alternative approach is based on a recent report of a common idiotype on MOG-specific autoantibodies in sera of guinea pigs affected with
  • This alternative approach involves immunomodulating the expression of the disease using anti-iodiotypic antibodies raised against anti-MOG IgGs purified by affinity
  • MOG peptides was synthesised according to the MOG cDNA sequence. Five (5) peptides of approximately 20 amino acids in length were initially synthesised. The MOG sequences to be synthesised
  • a peptide corresponding to amino acids 38-45 of MOG chemically coupled to KLH is first injected subcutaneously with 151 DEAE (as an adjuvant). Three weeks later, 80ug to 100ug of the same KLH-MOG peptide but emulsified with incomplete Freund's adjuvant are injected subcutaneously on the back of animals and then some weeks later 50ug of this peptide coupled to KLH is injected with incomplete Freund's adjuvant subcutaneously.
  • the animals are then reiejected into the footpads with either MOG peptide 35-55, MBP peptide 68-86, S100 protein and/or GFAP and/or with ovalbumin as controls, dissolved in saline and emulsified with an equal value of complete Freund's adjuvant (CFA) supplemented with 4mg per ml Mycobacterium tuberculosis (Difco H37RA).
  • CFA complete Freund's adjuvant
  • mice were injected intradermally in both hind foot pads with a total volume of 100 ul emulsion consisting of 200 ug of purified MOG or its peptides or 3 mg CNS homogenate dissolved in saline and emulsified with an equal volume of complete Freund's adjuvant (CFA) supplemented with 4 mg/ml Mycobacterium tuberculosis
  • CFA complete Freund's adjuvant
  • ear swelling assay 75ug of MOG peptides or 50ug of MOG was also used to assay T-cell responses to MOG.
  • MOG-specific T cell lines and clones- MOG-specific T cell lines and clones were generated in accordance with standard procedures.
  • MOG-specific T cell lines or clones generated from lymph nodes of animals immunized with encephalitogenic peptide(s) and/or the whole MOG molecule were injected intravenously (i.v.) into naive recipients at various cell concentrations
  • Control antibodies included anti-normal rat IgG and other antibodies
  • Each control and treatment group comprised 4-6 animals.
  • MBP degradation quantitated the extent of MBP degradation.
  • the extent of MBP degradation in the presence of control Mabs directed against plant proteins was not different from the basal degradation observed in the absence of added Igs and was the same as that observed in the presence of 4 different anti-myelin associated glycoprotein (MAG) Mabs, at least one of which is directed against an external epitope of MAG.
  • MAG anti-myelin associated glycoprotein
  • MOG is the primary target myelin antigen in at least some MS patients.
  • MOG is encephalitogenic strongly supports this postulate, and emphasizes the relevance of immune responses to MOG in MS.
  • Persistence of MOG reactivity together with reactivity to other myelin antigens points to a greater role for immune recognition of MOG in disease initiation with broadening of the autoimmune response to additional determinants on this and/or other autoantigens as the disease progresses.
  • Figure A Histological sections of the spinal cord showed multifocal demyelination, most prominent in the dorsal columns. Areas of demyelination were clearly demarcated from adjacent white matter. Within these areas, aggregates of mononuclear inflammatory cells were present around blood vessels. Cells with morphological features of monocyte/ macrophages infiltrated intact myelin adjacent to areas of demyelination.
  • Figure B Infiltration of residual myelin in dorsal column of cervical spinal cord by mononuclear cells with
  • FIG. 1 MBP degradation in human myelin incubated in the presence or absence of immunoglobulins (Igs) isolated from sera of affected rats.
  • Igs immunoglobulins
  • Each column represents the mean ⁇ SEM of percent MBP decrease in five separate myelin preparations incubated individually with Igs from control rats injected with CFA, with monoclonal anti- MOG Igs (8-18C5), with
  • MOG-EAE Igs from two different MOG- injected affected rats
  • MBP-EAE Igs from four rats with acute EAE induced with MBP
  • incubated in the absence of added Igs respectively.
  • Figure 2 Purified human MOG migrates as two 8-18C5-reactive bands on SDS-PAGE. Lanes 1 and 2, molecular weight markers (Pharmacia-LKB, Cat.No.17-0446-01) and purified human MOG (10ug) respectively, run on a 14% SDS mini-gel and stained with Coomassie Blue; lane 3, autoradiogram of purified human MOG (2ug) transferred onto PVDF membrane from a 14% SDS mini-gel and probed with monoclonal anti-MOG antibody
  • FIG. 1 Autolysis of purified human MOG. Autoradiogram of purified human MOG incubated at 37°C for various times (0, 0.5 hr, 1 hr and 3hrs), and immunoblotted using 125 I-labelled 8-18C5 (0.1 ug/ml) as the probe. The volume of the aliquots sampled at various times of incubation and run on a 14% SDS gel corresponded to a starting concentration of 5 ug MOG at 0 time.
  • Figure 4 - A Evidence of contamination with IgG in human MOG preparations after immunoaffinity chromatography on the
  • glycoprotein antibody followed by rabbit anti-mouse IgG and
  • Protein G chromatography (5 ug protein/lane); lane 2, unbound fraction from Protein G column (5 ug protein/lane); lane 3, bound fraction from Protein G column (5 ug protein/lane).
  • Lanes 1 and 2 represent molecular weight markers (MWs) and purified dMAG (3 ug) respectively, run on a 10% SDS gel and stained with Coomassie Blue; MWs shown are, from the top: phosphorylase b (94 kDa), albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (30 kDa). These markers are not relevant for the other antigens. Lanes 3-5 represent silber-stained PLP
  • Lane 6 shows purified human MOG (25 ug) run on a 15% gel; both 58 kDa and 28kDa MOG bands can be seen.
  • Lane 7 shows purified human MBP (5 ug) run on a 15% gel and which comprises the 18.5 kDa and the 17.3 kDa isoforms present in adult.
  • FIG. 6B Immunoblotting. Electrophoretograms of human myelin (30 ug/lane), purified human MOG (15 ug/lane), purified human MBP (1 ug/lane) or PLP (10 ug/lane) were transferred onto nitrocellulose or polyvinyleden difluoride membrane and probed as described in Material and Methods, with the various antibodies indicated.
  • the myelin lane was a positive control to ensure that the anti-PLP antibody used to probe the human MOG and MBP preparations would detect PLP contamination if present. It was probed with an anti-PLP antibody which does not recognize DM-20; in addition to the PLP band, higher and lower molecular weight forms were also detected.
  • FIG. 7 Sequence of human MOG.
  • the putative signal sequence is underlined with a dotted line and two predicted membrane spanning regions are underlined with a single line.
  • the potential N-linked glycosylation site is underlined with a double line and the possible poly adenylation signals are in bold.
  • the asterisk at 592 bp indicates where the
  • Figure 9 Amino acid sequence of the encephalitogenic and immunogenetic MOG peptide 35-55.
  • FIG. 10 N-terminal and internal fragment sequences of human MOG and comparison with known regions of rat and mouse MOG sequences. Amino acids identical in all three species are denoted by an asterisk; unidentified amino acids are shown as X.
  • Human and rat MOG was purified as previously reported (14). Lewis rats were injected in the hind foot pads with the indicated amount of MOG emulsified in CFA supplemented with Mycobacterium tuberculosis (4 mg/ml) and observed for 60 days. Rats were examined daily for clinical signs of disease and were graded as mild (loss of weight, hind leg weakness and/or balance impairment) or severe (mild disease plus hind and/or fore leg paralysis and/or severe balance impairment). Rats injected with 50 ⁇ g of guinea pig HBP were used as positive controls for EAE induction. CFA-injected animals served as negative controls. Serum antibody reactivity was determined by immunoblotting with whole brain homogenate, purified human myelin and purified MOG and MBP as antigens.
  • Lewis rats were injected as in Table 1 with the indicated amount of peptide( s) .
  • Anina ls were observed for 90 days and scored as in Table 1.
  • Rats inj ected with 50 ⁇ g of guinea pig MBP were used as positive controls. Serum antibody reactivity to peptides was determined by ELISA and to other myelin proteins by immunoblotting .
  • MOG and PLP 2/24 (8.3) 0/16(0) * P ⁇ 0.004 compared with control group.
  • ⁇ 2 values have one degree of freedom.
  • Kerlero de Rosbo N., Carnegie, P.R., Bernard, C.C.A., and Linthicum, D.S. (1984) Neurochem. Res. 9:1359-1369. Kerlero de Rosbo, N. and Bernard, C.C.A. J. Neurochem. (1989) 53:513-518.

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Abstract

La présente invention se rapporte à des procédés et à des compositions acceptables dans les domaines pharmaceutique et vétérinaire, convenant au traitement de la sclérose en plaques, une maladie auto-immune induite par lymphocytes et/ou lymphocytes B, chez les hommes ou les animaux, respectivement, par l'administration, à ces derniers, de: (i) la glycoprotéine d'oligodendrocytes de myéline (MOG); ou (ii) une protéine analogue à la MOG; ou (iii) une région immunodominante de MOG ou de la protéine analogue, en particulier un épitope immunodominant de MOG ou de la protéine analogue; ou (iv) un anticorps anti-idiotypique dirigé contre une proteíne ou un peptide de (i) à (iii); ou (v) des anticorps monoclonaux anti-idiotypiques dirigés contre une protéine ou un peptide de (i) à (iii). La présente invention se rapporte en outre à des agents diagnostiques, ainsi qu'à des procédés et des kits de détection de la sclérose en plaques ou d'autres maladies auto-immunes induites par lymphocytes T ou B chez les humains ou les animaux. La présente invention se rapporte également à des séquences nucléotidiques qui codent pour la MOG humaine ou une protéine humaine analogue à la MOG ou une région immunodominante de MOG humaine ou un épitope immunodominant de MOG humaine, ainsi qu'à la MOG humaine ou une protéine analogue à cette dernière ou une région immunodominante de MOG humaine ou un épitope immunodominant de MOG humaine, obtenus par des procédés standards de synthèse de protéines ou par des techniques de recombinaison d'ADN.
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